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| United States Patent |
5,336,783
|
|
Omura
, et al.
|
August 9, 1994
|
Calpain inhibitor cystamidin A and its production
Abstract
A novel calpain inhibitory active and low molecular weight substance,
cystamidin A and its production are provided. Cystamidin A of the
following formula
##STR1##
was produced by culturing Streptomyces sp. KP-1241 FERM BP-41 71 in a
nutrient medium and isolating the active substance from the cultured mass.
| Inventors:
|
Omura; Satoshi (Tokyo, JP);
Tanaka; Haruo (Tokyo, JP);
Shiomi; Kazuro (Tokyo, JP);
Liu; Jing R. (Beijing, CN)
|
| Assignee:
|
The Kitasato Institute (Tokyo, JP)
|
| Appl. No.:
|
057731 |
| Filed:
|
March 23, 1993 |
Foreign Application Priority Data
| Apr 20, 1992[JP] | 4-099666 |
| Feb 05, 1993[JP] | 5-019001 |
| Current U.S. Class: |
548/561 |
| Intern'l Class: |
C07D 207/323 |
| Field of Search: |
548/561
|
References Cited
U.S. Patent Documents
| 4376778 | Mar., 1983 | Ezaki et al. | 548/557.
|
| 5075452 | Dec., 1991 | Kirchlechner et al. | 548/561.
|
| Foreign Patent Documents |
| 3-157366 | Jul., 1991 | JP | 548/557.
|
| 4-095069 | Mar., 1992 | JP | 548/557.
|
Other References
"Darstellung von histaminahnlichen Substanze aus der pyrrolreihe",
Hoppe-Seyler's Zeitschrift fur Physiologische Chemie, Band 289, Heft 1,
1952, By Kutscher et al., pp. 229-233.
"The Role of Concepts in Structure-Activity Releationship Studies of
Opioid", Journal of Medicinal Chemistry, vol. 35, No. 11, 1992, By P.
Portoghese, pp. 2047-2054.
|
Primary Examiner: Lee; Mary C.
Assistant Examiner: McKane; Joseph K.
Attorney, Agent or Firm: Young & Thompson
Claims
What is claimed is:
1. Cystamidin A of the formula
##STR3##
2. Cystamidin A which has the following physico-chemical properties:
(1) Nature: white powder
(2) Molecular weight: 138.0793 (by mass spectrometric analysis)
(3) Molecular formula: C.sub.7 H.sub.10 N.sub.2 O
(4) Melting point: 146.degree.-148.degree. C.
(5) Ultraviolet absorption maximum (in methanol): 203 nm (.epsilon.=4400),
213 nm (shoulder, .epsilon.=4100), 260-280 nm (shoulder, .epsilon.=300)
(6) Infrared absorption maximum (KBr tablet): 3400, 3250, 3150, 1640, 1570,
1400, 1280, 1110, 970, 805, 740 cm.sup.-1
(7) Solubility in solvent: soluble in water, methanol, acetone, chloroform
and benzene
(8) Color reaction: positive for ninhydrin, Ebrlich and Rydon-Smith
reagents.
Description
FIELD OF THE INVENTION
This invention relates to a novel calpain inhibitor, cystamidin A, and to a
process for its production.
PRIOR ART
Calpain is a calcium-dependent cysteine protease which occurs in various
kinds of mammalian tissues. The enzyme is presumed to play a central role
in physiological or pathological events, and to be involved in the
activation of kinase-series enzymes such as protein kinase C and
phosphorylase kinase B by limiting proteolysis, and in the decomposition
of cytoskeletal proteins and of growth-factor- and hormone-receptors.
Diseases related to calpain are known. It is strongly suggested to be
involved in the turnover of muscle in muscular dystrophy, for example, and
in the disappearance of the Z line in skeletal muscle (Experimental
Medicine, 5:942 (1987)).
Furthermore, in a cell infected with human T-cell leukemia virus, extremely
increased activities of calpain and interleukin 2 receptor are observed.
This may be caused by an irregular reaction of cells to growth factor due
to alteration of receptor activity by an action of calpain on cytoskeletal
protein (Biochemistry, 57:1202 (1985)). Still further, calpain is thought
to have a relation to myocardial infarction (Experimental Medicine, 5:937
(1987)), demyelination (Modern Medicine, 43:81 3 (1988)) and inflammation
(ibid., 43:776 (1988)).
Calpastatin which is a specific inhibitory protein as to calpain, is known,
and is expected to be applicable as an effective therapeutic agent for
various excessive calpain-related syndromes. Calpastatin is, however, a
high molecular weight protein and hence it will be difficult to use as a
medicine. A low molecular weight calpain inhibitor is therefore desirable.
OBJECT OF THE INVENTION
An object of the present invention is therefore to provide a novel low
molecular weight calpain inhibitor and a process for its production.
SUMMARY OF THE INVENTION
It has now been found that a strain belonging to the genus Streptomyces
produces calpain inhibition in its cultured broth.
An object of the present invention is therefore to provide a novel low
molecular weight calpain inhibitor and a process for its production.
It has also been found that this calpain inhibition is due to a novel
compound cystamidin A of the formula
##STR2##
The invention also provides a process for the production of cystamidin A,
which comprises culturing a cystamidin A-producing microorganism belonging
to the genus Streptomyces, and isolating the thus-produced cystamidin A
from the cultured mass.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: UV spectrum of cystamidin A;
FIG. 2: IR spectrum of cystamidin A;
FIG. 3: .sup.1 H-NMR spectrum of cystamidin A; and
FIG. 4: .sup.13 C-NMR spectrum of cystamidin A.
DETAILED DESCRIPTION OF THE INVENTION
Cystamidin A of the present invention can be produced by culturing a
cystamidin A-producing microorganism belonging to the genus Streptomyces
in a nutrient medium, accumulating the cystamidin A in the medium, and
isolating the calpain inhibitor cystamidin A therefrom. The cystamidin
A-producing microorganism can be a cystamidin A-producing microorganism of
the genus Streptomyces and is not limited to the strain disclosed in this
specification. A preferred example of cystamidin A-producing microorganism
is Streptomyces sp. KP-1241 isolated from a soil sample collected in
China by the present inventors. The taxonomical properties of the strain
KP-1241 are set forth hereinbelow.
1. Morphological properties
The vegetative mycelia grow abundantly on both synthetic and complex agar
media and do not show fragmentation into coccoid forms or bacillary
elements. The aerial mycelia grow abundantly on inorganic salts-starch
agar and glycerol-asparagine agar to show a grayish color. The mature
sporophores form spiral spore chains and have more than 20 spores per
chain. The spores are oval in shape, 1.0 .mu.m.times.0.7 .mu.m in size and
have a spiny surface. Whorls, sclerotic granules, sporangia and
flagellated spores are not observed.
2. Cultural characteristics
The cultural characteristics are shown in Table 1. To investigate the
cultural characteristics and physiological properties of the strain, the
International Streptomyces Project (ISP) medium recommended by Shirling
and Gottlieb (Int. J. Syst. Bacteriol., 16:313-340 (1966)) was used. Color
Harmony Manual, 4th Ed., 1958 (Container Corporation of America, Chicago)
was used for color names and hue numbers. Cultures were observed after
incubation at 27.degree. C. for two weeks, if not otherwise specified.
TABLE 1
______________________________________
Cultural Characteristics of Strain KP-1241
Medium Cultural Characteristics
______________________________________
Sucrose-nitrate agar
G: poor, colorless
R: silver gray (3fe)
AM: poor, silver gray (3fe)
SP: none
Glucose-asparagine
G: good, light ivory (2ca)
agar (ISP) R: light wheat (2ea)
AM: abundant, silver gray (3fe)
SP: none
Glycerol-asparagine
G: good, colorless
agar (ISP) R: bamboo (2gc)
AM: abundant, silver gray (3fe)
SP: none
Inorganic salts-starch
G: good, light ivory (2ca)
agar (ISP) R: covert tan (2ge)
AM: abundant, dark covert gray (2ih)
SP: none
Tyrosine agar (ISP)
G: moderate, light ivory (2ca)
R: pale pink (3ca)
AM: poor, white (a)
SP: poor
Oatmeal agar (ISP)
G: moderate, colorless
R: silver gray (3fe)
AM: moderate, beige gray (3ih)
SP: none
Yeast extract-malt
G: good, colorless
extract agar (ISP)
R: mustard (21e)
AM: abundant, dark covert gray (2ih)
SP: none
Nutrient agar G: good, colorless
R: bisque (3ec)
AM: moderate, white (a)
SP: none
Peptone-yeast extract
G: moderate, light ivory (2ca)
iron agar (ISP)
R: honey gold (2ic)
AM: moderate, white (a)
SP: none
Glucose-nitrate
G: moderate, light mustard tan (2ie)
agar R: mustard (2le)
AM: poor, white (a)
SP: none
Glycerol-calcium
G: good, light ivory (2ca)
malate agar R: light ivory (2ca)
AM: poor, white (a)
SP: none
Glucose-peptone
G: moderate, bamboo (2gc)
R: bisque (3ec)
AM: moderate, white (a)
SP: amber (3pe)
______________________________________
Abbreviations:
G: growth of vegetative mycelium
R: reverse
AM: aerial mycelium
SP: soluble pigment
ISP: International Streptomyces Project
3. Physiological Properties
(+=active; -=inactive)
______________________________________
(1) Melanin formation
(a) Tyrosine agar +
(b) Peptone-yeast extract iron agar
+
(c) Glucose-peptone-gelatin medium
-
(21-23.degree. C.)
(d) Tryptone-yeast liq. -
(2) Tyrosinase reaction +
(3) H.sub.2 S production -
(4) Nitrate reduction +
(5) Liquefaction of gelatin (21-23.degree. C.)
+
(6) Hydrolysis of starch +
(7) Coagulation of milk (27.degree. C.)
-
(8) Peptonization of milk (27.degree. C.)
+
(9) Temperature range for growth 12-34.degree. C.
Optimum temperature range for growth 24.degree. C.
(10) Utilization of carbon sources
(Pridham and Gottlieb agar medium)
Utilized: D-glucose, D-mannitol, D-fructose,
i-Inositol;
Weakly utilized: L-arabinose, D-rhamnose;
Not utilized: D-xylose, raffinose, melibiose, sucrose.
(11) Cellulolytic activity
______________________________________
4. Chemical composition
The DAP (diaminopimelic acid)-isomer in the cell wall of strain KP-1241 is
determined to be LL-type.
The taxonomical properties of the strain KP-1241 are illustratively
summarized as follows. The DAP-isomer in the cell wall is LL-type. The
aerial mycelia form spiral spore chains with smooth surfaces. As to the
cultural characteristics, the vegetative mycelia show white or gray on
various media. Soluble pigment is slightly produced in tyrosine agar and
glucose-peptone agar.
From the taxonomic properties described above, strain KP-1241 is considered
to be the white or gray series of the genus Streptomyces according to the
classification by Pridham and Tresner (Bergey's Manual of Determinative
Bacteriology, Vol. 8, pp. 748-829, 1974). The strain is referred to as
genus Streptomyces and the species is designated Streptomyces sp. KP-1241.
The strain was deposited in the National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology, Japan, under the
name Streptomyces sp. KP-1241 and the accession No. is FERM BP-41 71.
In the present invention, the above strain is merely illustrative, and the
above strain, its mutants and other cystamidin A-producing microorganisms
belonging to the genus Streptomyces can naturally be used. The calpain
inhibitor, cystamidin A of the present invention can be produced by
inoculating and culturing aerobically the above strain, for example, in a
nutrient medium suitable for cystamidin A production. A nutrient source
such as any nutrient medium for Streptomyces can be used.
Commercially available nitrogen sources such as peptone, meat extract, corn
steep liquor, cotton seed meal, peanut meal, soybean meal, yeast extract,
NZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate and
ammonium sulfate, and carbon sources such as glycerin, starch, glucose,
galactose, lactose and mannose can be used. Furthermore, carbon sources
such as fatty substances, and inorganic salts such as sodium chloride,
phosphate salts, calcium carbonate and magnesium sulfate can also be used.
The other essential trace metallic salt and anti-foam agents such as
animal, vegetable or mineral oil can be added to the medium if required.
These nutrient sources and other additives useful for the production of
cystamidin A can be used, which is to say that any cultural material for
Streptomyces can be used in the present invention. Liquid culture such as
submerged aeration culture is preferable for quantity production of
cystamidin A. The temperature for culturing the microorganisms can be
adjusted to grow the strain within a range of suitable production of
cystamidin A. Culturing conditions can be selected and controlled for the
production of cystamidin A, depending upon the nature of the production
microorganisms.
Cystamidin A is mainly produced in a cultured broth. Crude cystamidin A can
be isolated by extracting the cultured filtrate with a water-immiscible
organic solvent such as butanol, or by eluting the absorbed substance in
an absorption resin with a water-containing organic solvent. In addition
to the above extraction procedure, the isolation method used for low
molecular weight substances such as absorption chromatography,
gel-filtration chromatography, preparative thin layer chromatography,
countercurrent partition chromatography and high performance liquid
chromatography, and preferably combinations and repetitions thereof, can
be used for isolating and obtaining pure cystamidin A.
The physico-chemical properties of cystamidin A are shown by the following:
1. Nature: white powder
2. Molecular weight: 138.0793 (by mass spectrometric analysis)
3. Molecular formula: C.sub.7 H.sub.10 N.sub.2 O
4. Melting point: 146.degree.-148.degree. C.
5. Ultraviolet absorption maximum (in methanol): (FIG. 1) 203 nm
(.epsilon.=4400), 213 nm (shoulder, .epsilon.=4100), 260-280 nm (shoulder,
.epsilon.=300)
6. Infrared absorption maximum (KBr tablet): (FIG. 2) 3400, 3250, 3150,
1640, 1570, 1400, 1280, 1110, 970, 805, 740 cm.sup.-1
7. .sup.1 H-NMR spectrum (in DMSO-d.sub.6, ppm): (FIG. 3) 10.40 br. s (1H),
7.24 br. s (1H), 6.69 br. s (1H), 6.60 dd (1H), 6.50 dd (1H), 5.86 dd
(1H), 2.59 t (2H), 2.25 t (2H)ppm (s=singlet, d=doublet, t=triplet,
br=broad)
8. .sup.13 C-NMR spectrum (in DMSO-d.sub.6, ppm): (FIG. 4) 174.3 s, 121.8
s, 117.3 d, 114.6 d, 107.5 d, 37.0 t, 22.8 t
9. Solubility in solvent: soluble in water, methanol, acetone, chloroform
and benzene
10. Color reaction: positive for ninhydrin, Ebrlich and Rydon-Smith
reagents
From the above physico-chemical properties and the spectra data, the
structure of cystamidin A was elucidated as shown in the formula (1)
hereinbefore.
As illustrated in detail by the physico-chemical properties of cystamidin
A, the substance has never been known and reported and is a novel
compound.
The biological properties of cystamidin A are illustrated as follows:
The inhibitory activity of cystamidin A against calpain using casein as
substrate according to the method by Saito et al. (Agr. Biol. Chem.,
51:361 (1987)), IC.sub.50 (50% inhibition) is 0.20 .mu.g/ml.
Cystamidin A shows no antimicrobial activity at 100 .mu.g/disk (paper disk
method) against various kinds of bacteria, yeast and fungi. The IC.sub.50
value against B-16 melanoma cells in vitro was more than 25 .mu.g/ml. The
LD.sub.50 (ip) of cystamidin A in mice is >200 mg/kg.
Effect of the Invention
As explained hereinabove, the novel compound cystamidin A is a low
molecular weight substance and shows strong inhibitory action on calpain.
Therefore it can be used not only as a reagent but also as a
pharmaceutical.
EXAMPLES
The present invention will be illustrated by the following examples, which
are, however, not to be construed as limiting.
EXAMPLE 1
A loopful of mycelia of a stock culture of Streptomyces sp. KP-1241 FERM
BP-4171 was transferred into each of 14 flasks each of which was a 500-ml
Erlenmeyer flask containing 100 ml of a medium consisting of glucose 0.1%,
soluble starch (Kanto Chem. Co.) 2.4%, peptone (Kyokuto Pharmaceutical
Industrial Co.) 0.3%, meat extract (Kyokuto Pharmaceutical Industrial Co.)
0.3%, yeast extract (Oriental Yeast Co.) 0.5% and CaCO.sub.3 0.4% (pH 7.0
before sterilization), and the mixture was then incubated on a rotary
shaker at 27.degree. C. for 3 days to give a seed culture. The seed
culture (1.4 lit.) was inoculated into a tank fermenter containing 70
liters of a medium consisting of glycerol 2.0%, soybean meal 2.0% and NaCl
1 0.3% (pH 7.0 before sterilization), and the medium was incubated with
agitation at 200 rpm and aeration at the rate of 35 lit./min. at
27.degree. C. for 4 days.
The cultured broth was centrifuged and the supernatant was charged on a
column of Diaion HP20 (5 lit., Mitsubishi Chem. Ind. Ltd.) and eluted with
20% methanol. The eluate (25 lit.) showing calpain inhibitory activity was
extracted twice with ethyl acetate (18 lit.) The extract was concentrated
in vacuo to obtain a brownish oil (55 g). This oil was dissolved in water
(1.5 lit.), introduced onto a reversed phase silica gel column (1000 ml,
YMC* GEL ODS-AQ 120-S50, YMC Co.) packed with 50% methanol, and developed
with 20% methanol.
The active fractions were collected and concentrated in vacuo to obtain
another brownish oil (7.2 g). The latter oil was dissolved in a small
volume of methanol, and the solution was mixed with silica gel powder (50
g, Merck Art. 7734), then methanol was stripped from the mixture under
reduced pressure. The dried silica gel was introduced onto a column of
silica gel (210 g, Merck Art. 7734) and eluted with a mixture of
chloroform and methanol (30:1). The active fraction was collected and
evaporated under reduced pressure to give still another brownish oil (439
mg).
The last oil was dissolved in a small volume of methanol and applied on
HPLC (Capcell Pak C18, SG120, .phi.20 .times.250 mm, Shiseido Co.)
separately 12 times, with 5% acetonitrile at a flow rate of 8 ml/min.,
checking and detecting the absorption at 205 nm. The peak showing calpain
inhibitory activity, eluted for 17 min., was collected. The extract was
concentrated under reduced pressure to obtain a white powder of cystamidin
A (370 mg).
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